SUMMARY. Mycobacterium kansasii (Mk) is among the six most common nontuberculous mycobacterial pathogens associated with opportunistic pulmonary infections in the United States and many other areas of the world, where it is often surpassed only by the Mycobacterium avium complex. Mk causes a chronic pulmonary disease similar to tuberculosis (TB). Mk infections continue to rise with the uncurbed HIV epidemic and the increase of other predisposing factors in populations around the world. Alarmingly, the juxtaposition of the HIV and TB epidemics creates fertile grounds for the rise of drug resistant Mk strains, and Mk isolates resistant to anti-TB drugs that are also a cornerstone of Mk infection treatments have been documented. Drug resistance is a threat to the future control of Mk infections, which currently requires long-term, multidrug courses with adverse side effects and challenging compliance. Worrisomely, Mk biology remains essentially unexplored. The scarcity of gene function studies in Mk and the lack of defined Mk mutants are major barriers to progress in the field. Mk-focused gene function studies are required to develop a comprehensive understanding of Mk biology, which is critical to better position the community to address the public health challenge arising from Mk infections. A deeper knowledge of Mk biology will help illuminate potential avenues to better therapeutics against Mk infections. Understanding Mk biology is also necessary to furthering preparedness against the inevitable upcoming of multidrug resistant Mk strains as a collateral outcome of the pervasive use of anti-TB drugs to curb the TB epidemic. Dissection of Mk biology may also help us understand the biology of other mycobacteria. The long term goal set for our Mk research is to apply genetic approaches to interrogate Mk biology. The underlying central hypothesis behind this R03 project is that transposon (Tn) mutagenesis-based genetic screens are powerful approaches to dissect gene function and unveil genotype-phenotype associations in Mk. To begin testing this hypothesis, the two following Specific Aims are proposed. (1) Construction of a genome-wide, near-saturation, arrayed library of Tn mutants of Mk. (2) Screen for and genetic characterization of colonial morphology mutants of Mk. The reason to select this screen at the onset is that it will lead to identification of a mutant population enriched in isolates with insertions in genes involved in defining properties of the cell envelope, which is a critical player in the ability of mycobacteria to produce disease and contributes to the mycobacterial resilience to drug treatments and host defenses. In all, this R03 proposal focuses on an underexplored important topic and rests on a foundation of solid preliminary data, feasible approaches, and an experienced Principal Investigator uniquely positioned to pursue the project. The project will deliver new knowledge to the field of Mk gene function, develop a first Mk mutant library, generate a pipeline of defined mutants for future virulence studies, validate genetic tools for Mk, and build research capacity for additional future genetic screens, some powered by combination of saturation Tn mutagenesis and deep sequencing.